Biology subjects

Bareke, E.

Publications and source records attributed to Bareke, E..

12 recordsLinked to original sources

bioRxiv2026

Developmental pleiotropy revealed by mosaic heterozygous Snrpb deletion underlies CCMS-like axial skeletal defects

Cerebro-Costo-Mandibular Syndrome (CCMS) is a rare congenital disorder due to pathogenic variants in the core spliceosomal gene SNRPB. Affected individuals present with axial skeletal abnormalities, including cleft palate, micrognathia, posterior rib gaps, and a bell-shaped thorax. The molecular basis of these axial defects remains poorly understood, limiting the development of targeted interventions. To study the temporal and molecular requirements of SNRPB during axial development, we generated a mouse model of CCMS using an inducible Cre-lox system to conditionally delete Snrpb. Mosaic deletion of exons 2-3 of Snrpb after gastrulation resulted in the full spectrum of CCMS-like abnormalities, including micrognathia, posterior rib gaps and a reduced thoracic cavity. Despite normal somite morphology and patterning, transcriptomic analysis of E9.5 mutant somites revealed upregulation of p53 pathway genes and mis-expression of retinoic acid (RA) signaling components, consistent with reduced RA signaling. Alternatively spliced genes in Snrpb mutant somites were associated with post-transcriptional regulation, including chromatin modifiers. To test if RA pathway supplementation can rescue axial defects, we performed dietary RA supplementation; however, this failed to rescue mutant phenotypes, suggesting that p53 activation and chromatin dysregulation additionally contribute to disease pathogenesis. Together, these findings establish the first in vivo model of CCMS-associated axial skeletal abnormalities and indicate that Snrpb dysfunction disrupts the early specification of axial skeletal identity without overtly altering somite patterning.

developmental biology↗

bioRxiv2025

Integrated Proteomic and Epigenomic Analysis Reveals IGF2 as a Vulnerability in PRC2-Deficient Malignant Peripheral Nerve Sheath Tumors

Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive sarcomas with limited therapeutic options. Loss of the Polycomb repressive complex 2 (PRC2), via inactivating mutations in SUZ12 or EED, occurs frequently in MPNSTs and is associated with poor prognosis. However, the downstream chromatin and signaling consequences of these mutations remain incompletely understood. Here, we show that PRC2 deficiency in MPNST cells induces coordinated chromatin remodeling, characterized by loss of repressive H3K27me3 and gain of activating marks, including H3K27ac and H3K36me2. Integrative epigenomic, transcriptomic, and proteomic profiling revealed that this chromatin reprogramming activates a fetal-like growth signature centered on insulin-like growth factor 2 (IGF2) and its post-transcriptional regulators, Insulin-like Growth Factor 2 mRNA-Binding Protein (IGF2BP1-3). Functional studies demonstrate that PRC2-deficient cells are selectively dependent on IGF2 for proliferation, and that restoration of SUZ12 suppresses IGF2 expression and reduces growth. Analysis of human MPNST tumors confirms upregulation of the IGF2-IGF2BP axis in PRC2-deficient tumors, highlighting its clinical relevance. Together, these findings link PRC2 loss to activation of fetal growth factor-driven oncogenic signaling and identify IGF2 and its regulatory network as potential vulnerabilities in this aggressive tumor subtype.

cancer biology↗

bioRxiv2025

Cellular Reprogramming of H3K27M Pediatric High-Grade Glioma to Neuron-like State

This study explores the cell fate reprogrammability of H3K27M-mutant pediatric high-grade gliomas (pHGG) using neuronal transdifferentiation as a potential targeted therapy. We treated the BT245 patient-derived glioma cell line with pharmacological combinations targeting neuronal differentiation pathways and performed bulk RNA sequencing to characterize gene expression patterns driving cell fate transitions. Our findings reveal that the drug combinations induce transcriptomic changes consistent with differentiation towards neuronal phenotypes, including the upregulation of synaptic and dendritic signaling genes and the downregulation of malignant signatures. In comparison, astrocytic differentiation media (DM) and H3K27M knockout (KO) promote residual astrocytic phenotypes, suggesting neuronal transdifferentiation as a more effective strategy for mitigating tumor aggressiveness and progression. Differentially expressed genes such as GRIK1, GRIN1, NRXN3, NRXN1, CALB2, SCGN, SLC32A1, SLC1A2, KCNC3, and neurodevelopmental regulators including WNT7A, DLX6, ERBB4, ARX, BCL11B, SEMA3C, and FGFBP3 were identified as key markers regulating the neuron-like lineage transition. This study demonstrates that pHGGs can be phenotypically redirected toward neuronal-like identities through modulating cell fate differentiation programs. These findings advance the concept of differentiation therapy as a promising intervention to reduce phenotypic plasticity and malignancy in pHGG ecosystems. While these are early in vitro findings, the ability to steer and control glioma cells toward stable, less malignant fates offers promising translational potential for patient-centered targeted therapies.

cancer biology↗

bioRxiv2025

ATR and PKMYT1 inhibition re-sensitize a subset of TNBC patient-derived models to carboplatin inducing mitotic catastrophe

Triple negative breast cancer (TNBC) is associated with poor prognosis and is mainly treated with chemotherapy-based regimens, often including carboplatin. Resistance to carboplatin is a common clinical issue that is either initially present or develops with treatment. Overcoming this resistance is a significant clinical challenge, which highlights the need for novel therapeutic strategies. We used a pooled shRNA screening approach with a chemoresistant TNBC patient-derived xenograft (PDX) cell (PDXC) line to identify targets whose knockdown would enhance the efficacy of carboplatin. This screening led to the identification of the ATR (ataxia telangiectasia and Rad3-related) gene as a key therapeutic vulnerability. Inhibiting ATR with BAY1895344 or AZD6738 re-sensitized carboplatin-resistant PDXCs and PDXs to carboplatin, resulting in an increase in DNA damage, and apoptosis. ATR inhibition disrupts the dependence of carboplatin-resistant cells on the S and G2/M checkpoints for DNA repair, leading to mitotic catastrophe. We further found that the addition of ATR inhibitors to carboplatin reversed a FOXM1-targeted gene program enabling premature passage into mitosis. Moreover, targeting PKMYT1, a regulator of cyclin-dependent kinase 1 (CDK1) controlling the G2/M checkpoint, through knockdown or with the novel PKMYT1 inhibitor RP-6306, also enhanced carboplatin efficacy in our TNBC PDXC. Molecular factors associated with response to the ATR inhibitor/carboplatin combination included low RNA levels of PKMYT1. These results underscore the pivotal roles of ATR and PKMYT1 in mediating resistance to carboplatin in TNBC and support targeting these pathways to overcome carboplatin resistance in this disease.

cancer biology↗

bioRxiv2024

ChIPbinner: An R package for analyzing broad histone marks binned in uniform windows from ChIP-Seq or CUT&RUN/TAG data

BackgroundThe decreasing costs of sequencing, along with the growing understanding of epigenetic mechanisms driving diseases, have led to the increased application of chromatin immunoprecipitation (ChIP-Seq), Cleavage Under Targets & Release Using Nuclease (CUT&RUN) and Cleavage Under Targets and Tagmentation (CUT&TAG) sequencing - which are designed to map DNA or chromatin-binding proteins to their genome targets - in biomedical research. Existing software tools, namely peak-callers, are available for analyzing data from these technologies, although they often struggle with diffuse and broad signals, such as those associated with broad histone post-translational modifications (PTMs). ResultsTo address this limitation, we present ChIPbinner, an open-source R package tailored for reference-agnostic analysis of broad PTMs. Instead of relying on pre-identified enriched regions from peak-callers, ChIPbinner divides (bins) the genome into uniform windows. Thus, users are provided with an unbiased method to explore genome-wide differences between two samples using scatterplots, principal component analysis (PCA), and correlation plots. It also facilitates the identification and characterization of differential clusters of bins, allowing users to focus on specific genomic regions significantly affected by treatments or mutations. We demonstrated the effectiveness of this tool through a case study assessing H3K36me2 depletion following NSD1 knockout in head and neck squamous cell carcinoma, highlighting the advantages of ChIPbinner in detecting broad histone mark changes over existing software. ConclusionsBinned analysis provides a more holistic view of the genomic landscape, allowing researchers to uncover broader patterns and correlations that may be missed when solely focusing on individual peaks. ChIPbinner offers researchers a convenient tool to perform binned analysis. It improves on previously published software by providing a clustering approach that is independent of each bins differential enrichment status, while also offering additional features for downstream analysis of these differentially enriched bins.

bioinformatics↗

bioRxiv2024

H3K36 Methylation - a Guardian of Epigenome Integrity

H3K36 methylation is emerging as a key epigenetic modification with strong implications in genetic disease and cancer. However, the mechanisms through which H3K36me impacts the epigenome and asserts its functional consequences are far from understood. Here, we use mouse mesenchymal stem cell lines with successive knockouts of the H3K36 methyltransferases: NSD1, NSD2, SETD2, NSD3, and ASH1L, which result in progressive depletion of H3K36me and its complete absence in quintuple knockout cells, to finely dissect the role of H3K36me2 in shaping the epigenome and transcriptome. We show that H3K36me2, which targets active enhancers, is important for maintaining enhancer activity, and its depletion results in downregulation of enhancer-dependent genes. We demonstrate the roles of H3K36me2/3 in preventing the invasion of gene bodies by the repressive H3K27me modifications. Finally, we observe a previously undescribed relationship between H3K36me and H3K9me3: Following the depletion of H3K36me2, H3K9me3 is redistributed away from large heterochromatic domains and towards euchromatin. This results in a drastic decompartmentalization of the genome, weakening the boundaries between active and inactive compartments, and a catastrophic loss of long-range inter-compartment interactions. By studying cells totally devoid of H3K36 methyltransferase activity, we uncover a broad range of crucial functions of H3K36me in maintaining epigenome integrity.

genomics↗

bioRxiv2024

Etiology of Craniofacial and Cardiac Malformations in a Mouse Model of SF3B4-Related Syndromes

Pathogenic variants in SF3B4 are responsible for the acrofacial disorders Nager and Rodriguez Syndrome, also known as SF3B4-related Syndromes, associated with malformations in the head, face, limbs, vertebrae as well as the heart. To uncover the etiology of craniofacial malformations found in SF3B4-related syndromes, mutant mouse lines with homozygous deletion of Sf3b4 in neural crest cells (NCC) were generated. Like in human patients, these embryos had craniofacial and cardiac malformations with variable expressivity and penetrance. The severity and survival of Sf3b4 NCC mutants was modified by the level of Sf3b4 in neighbouring non NCC. RNA sequencing analysis of heads of embryos prior to morphological abnormalities showed significant changes in expression of genes forming the NCC regulatory network, as well as an increase in exon skipping. We identified several key transcription factors and histone modifiers involved in craniofacial and cardiac development with increased exon skipping. Increased exon skipping was also associated with use of a more proximal branch point, as well as an enrichment in thymidine bases in the 50bp around the branch points. We propose that decrease in Sf3b4 causes changes in the expression and splicing of transcripts required for proper craniofacial and cardiac development, leading to abnormalities.

developmental biology↗

bioRxiv2023

Systematic perturbations of SETD2, NSD1, NSD2, NSD3 and ASH1L reveals their distinct contributions to H3K36 methylation

BackgroundMethylation of histone 3 lysine 36 (H3K36me) has emerged as an essential epigenetic component for the faithful regulation of gene expression. Despite its importance in development, disease, and cancer, how the molecular agents collectively shape the H3K36me landscape is unclear. ResultsWe use a mouse mesenchymal stem cell model to perturb the H3K36me deposition machinery and infer the activities of the five most prominent players: SETD2, NSD1, NSD2, NSD3, and ASH1L. We find that H3K36me2 is the most abundant of the three methylation states and is predominantly deposited at intergenic regions by NSD1, and partly by NSD2. In contrast, H3K36me1/3 are most abundant within exons and are positively correlated with gene expression. We demonstrate that while SETD2 deposits most H3K36me3, it also deposits H3K36me2 within transcribed genes. Additionally, loss of SETD2 results in an increase of exonic H3K36me1, suggesting other H3K36 methyltransferases (K36MTs) prime gene bodies with lower methylation states ahead of transcription. Through a reductive approach, we uncover the distribution patterns of NSD3- and ASH1L-catalyzed H3K36me2. While NSD1/2 establish broad intergenic H3K36me2 domains, NSD3 deposits H3K36me2 peaks on active promoters and enhancers. Meanwhile, the activity of ASH1L is restricted to the regulatory elements of developmentally relevant genes, and our analyses implicate PBX2 as a potential recruitment factor. ConclusionsWithin genes, SETD2 deposits both H3K36me2/3, while the other K36MTs are capable of depositing H3K36me1/2 independently of SETD2 activity. For the deposition of H3K36me1/2, we find a hierarchy of K36MT activities where NSD1>NSD2>NSD3>ASH1L. While NSD1 and NSD2 are responsible for most genome-wide propagation of H3K36me2, the activities of NSD3 and ASH1L are confined to active regulatory elements.

genomics↗

bioRxiv2021

Snrpb, the gene mutated in CCMS, is haploinsufficient and required for proper morphogenesis and splicing

Heterozygous mutations in SNRPB, an essential core component of the five small ribonucleoprotein particles of the spliceosome, are responsible for Cerebrocostomandibular Syndrome (CCMS). We show that Snrpb heterozygous embryos arrest shortly after implantation. Additionally, heterozygous deletion of Snrpb in the developing brain and neural crest cells models craniofacial malformations found in CCMS, and results in death shortly after birth. RNAseq analysis of mutant heads prior to morphological defects revealed increased exon-skipping and intron-retention in association with increased 5 splice site strength. We found increased exon-skipping in negative regulators of the P53-pathway, increased levels of nuclear P53, P53-target genes. However, removing TrP53 in Snrpb heterozygous mutant neural crest cells did not completely rescue craniofacial development. We also found a small but significant increase in exon-skipping of several transcripts required for head and midface development, including Smad2 and Rere. Furthermore, mutant embryos exhibited ectopic or missing expression of Fgf8 and Shh, which are required to coordinate face and brain development. Thus, we propose that mis-splicing of transcripts that regulate P53-activity and craniofacial-specific genes both contribute to craniofacial malformations.

developmental biology↗

bioRxiv2020

Mis-splicing of Mdm2 leads to increased P53-activity and craniofacial defects in a MFDM Eftud2 mutant mouse model

EFTUD2, a GTPase and core component of the splicesome, is mutated in patients with mandibulofacial dysostosis with microcephaly (MFDM). We generated a mutant mouse line with conditional mutation in Eftud2 and used Wnt1-Cre2 to delete it in neural crest cells. Homozygous deletion of Eftud2 leads to neural crest cell death and malformations in the brain and craniofacial region of embryos. RNAseq analysis of embryonic mutant heads revealed a significant increase in exon skipping, in retained introns and enriched levels of Mdm2 transcripts lacking exon 3. Mutants also had increased nuclear P53, higher expression of P53-target genes, and increased cell death. Their craniofacial development was significantly improved when treated with Pifithrin-, an inihibitor of P53. We propose that craniofacial defects caused by mutations of EFTUD2 are a result of mis-splicing of Mdm2 and P53-associated cell death. Hence, drugs that reduce P53 activity may help prevent craniofacial defects associated with spliceosomopathies.

developmental biology↗

bioRxiv2020

TDP-43 stabilizes transcripts encoding stress granule protein G3BP1: potential relevance to ALS/FTD

TDP-43 nuclear depletion and concurrent cytoplasmic accumulation in vulnerable neurons is a hallmark feature of progressive neurodegenerative proteinopathies such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Cellular stress signalling and stress granule dynamics are now recognized to play a role in ALS/FTD pathogenesis. Defective stress granule assembly is associated with increased cellular vulnerability and death. G3BP1 (Ras-GAP SH3-domain-binding protein 1) is a critical stress granule assembly factor. Here, we define that TDP-43 stabilizes G3BP1 transcripts via direct binding of a highly conserved cis regulatory element within the 3UTR. Moreover, we show in vitro and in vivo that nuclear TDP-43 depletion is sufficient to reduce G3BP1 protein levels. Finally, we establish that G3BP1 transcripts are reduced in ALS/FTD patient neurons bearing TDP-43 cytoplasmic inclusions/nuclear depletion. Thus, our data suggest that, in ALS/FTD, there is a compromised stress granule response in disease-affected neurons due to impaired G3BP1 mRNA stability caused by TDP-43 nuclear depletion. These data implicate TDP-43 and G3BP1 loss of function as contributors to disease.

neuroscience↗

bioRxiv2020

Epigenome dysregulation resulting from NSD1 mutation in head and neck squamous cell carcinoma

Epigenetic dysregulation has emerged as an important mechanism of oncogenesis. To develop targeted treatments, it is important to understand the epigenetic and transcriptomic consequences of mutations in epigenetic modifier genes. Recently, mutations in the histone methyltransferase gene NSD1 have been identified in a subset of head and neck squamous cell carcinomas (HNSCCs) - one of the most common and deadly cancers. Here, we use whole (epi)genome approaches and genome editing to dissect the downstream effects of loss of NSD1 in HNSCC. We demonstrate that NSD1 mutations are directly responsible for loss of intergenic H3K36me2 domains, followed by loss of DNA methylation, and gain of H3K27me3 in the affected genomic regions. We further show that those regions are enriched in cis-regulatory elements and that subsequent loss of H3K27ac correlates with reduced expression of their target genes. Our analysis identifies genes and pathways affected by the loss of NSD1 and paves the way to further understanding the interplay among chromatin modifications in cancer.

genomics↗